Reconstruction and Analysis of the Immune-Related LINC00987/A2M Axis in Lung Adenocarcinoma

Abstract

Enhancer RNAs (eRNAs) participate in tumor growth and immune regulation through complex signaling pathways. However, the immune-related function of the eRNA-mRNA axis in lung adenocarcinoma (LUAD) is unclear. Data on the expression of eRNAs and mRNAs were downloaded from The Cancer Genome Atlas, GEO, and UCSC Xena, including LUAD, and pan-cancer clinical data and mutational information. Immune gene files were obtained from ImmLnc and ImmPort databases. Survival indices, including relapse-free and overall survival, were analyzed using the Kaplan-Meier and log-rank methods. The level of immune cell infiltration, degree of tumor hypoxia, and tumor cell stemness characteristics were quantified using the single-sample gene set enrichment analysis algorithm. The immune infiltration score and infiltration degree were evaluated using the ESTIMATE and CIBERSORT algorithms. The tumor mutation burden and microsatellite instability were examined using the Spearman test. The LUAD-associated immune-related LINC00987/A2M axis was down-regulated in most cancer types, indicating poor survival and cancer progression. Immune cell infiltration was closely related to abnormal expression of the LINC00987/A2M axis, linking its expression to a possible evaluation of sensitivity to checkpoint inhibitors and response to chemotherapy. Abnormal expression of the LINC00987/A2M axis was characterized by heterogeneity in the degree of tumor hypoxia and stemness characteristics. The abnormal distribution of immune cells in LUAD was also verified through pan-cancer analysis. Comprehensive bioinformatic analysis showed that the LINC00987/A2M axis is a functional and effective tumor suppressor and biomarker for assessing the immune microenvironment and prognostic and therapeutic evaluations of LUAD.

Keywords: LINC00987/A2M axis; LUAD; eRNA; immune cell infiltration; tumor cell stemness; tumor hypoxia.

FIGURE 1

Workflow. Workflow for the identification and verification of the immune-related LINC00987/A2M axis in LUAD and pan-cancer.

FIGURE 2

Identification of clinical characteristics of the two immune-related genes in the LUAD TCGA cohort. Differential expression of LINC00987 (A–D) and A2M (E–H) among the T (tumor), N (node), M (metastasis), and stage, evaluated using the Wilcoxon test.

FIGURE 3

Correlation of overall (OS) and relapse-free survival (RFS) with LINC00987 and A2M in datasets. (A–D) OS and RFS curves for LINC00987 and A2M in the TCGA cohort. (E–H) OS and RFS curves for A2M in the GSEnew and GSE31210 cohorts. (I,J) OS and RFS curves for combinations of LINC00987 and A2M in the TCGA cohort.

FIGURE 4

Distribution of immune-related characteristics in different subgroups related to LINC00987 and A2M in the TCGA and GEO datasets. (A,B) Heatmap of the expression of LINC00987 and A2M and 29 immune-related gene sets generated by ssGSEA in TCGA cohort. (C,D) Heatmap of the expression of A2M and 29 immune-related gene sets generated by ssGSEA in two GEO cohorts. Violin-plot showing the difference in ImmuneScore, StromalScore, ESTIMATEScore, and TumorPurity in (E) LINC00987 and (F–H) A2M subgroups in TCGA and GEO cohorts. *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.

FIGURE 5

Differences in the distribution of 22 human immune cells in LINC00987 and A2M expression groups estimated using the CIBERSORT algorithm. Boxplots for (A) LINC00987 and (B) A2M in the TCGA cohort. Boxplots for A2M in the (C) GSE31210 and (D) GSEnew cohorts. *p < 0.05, **p < 0.01, and ***p < 0.001.

FIGURE 6

Gene sets functional enrichment analysis. (A) GSEA of high vs. low expression of LINC00987. (B) GSEA of high vs. low expression of A2M in the TCGA cohort. Correlation of the expression of (C) LINC00987 and (D) A2M with hypoxia gene sets. Correlation of the expression of (E) LINC00987 and (F) A2M with tumor stem cell characteristics. *p < 0.05, ***p < 0.001, and ****p < 0.0001.

FIGURE 7

Response to immunotherapy and sensitivity to chemotherapy in relation to the expression of LINC00987 and A2M in TCGA, GSE31210, and GSEnew cohorts. (A) Sensitivity response of high vs. low expression of LINC00987 to PD1 and CTLA-4 inhibitors (p = 0.01). (B) Boxplots of the estimated IC50 for paclitaxel in high vs. low expression of LINC00987/A2M.

FIGURE 8

Lego plot representation of mutation patterns in LUAD samples in relation to the LINC00987/A2M axis. Single-nucleotide substitutions are divided into six categories with 16 surrounding flanking bases. The pie chart in the upper left shows the proportion of six categories of mutation patterns.

FIGURE 9

Levels of expression and Kaplan-Meier survival analysis of LINC00987 and A2M in pan-cancer analysis. (A,B) Population differences in the expression of LINC00987 and A2M. (C,D) Pairing differences in the expression of LINC00987 and A2M. (E,F) Red values indicate statistical significance of the p value (p < 0.05). HR hazard ratio, 95% CI 95% confidence interval, *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.

FIGURE 10

Correlation of alterations in TMB and MSI with the level of expression of LINC00987 and A2M in various tumors. (A,C) Radar chart showing the correlation between LINC00987 and TMB (A) and MSI (C) in 33 cancer types. (B,D) Relationship between A2M and TMB (B) and MSI (D). *p < 0.05, **p < 0.01, and ***p < 0.001.

FIGURE 11

Immune regulation according to the expression of LINC00987 and A2M in the pan-cancer analysis. (A) Correlation between the level of expression of LINC00987 and A2M and the immune score in multiple tumors determined by ESTIMATE. (B) Correlation between the expression of LINC00987/A2M and infiltration by 22 types of immune cells in pan-cancer analysis. Red indicates a correlation coefficient > 0, whereas blue indicates a correlation coefficient < 0. p < 0.05.

FIGURE 12

Schematic diagram. LUAD patients with a high LINC00987/A2M axis expression have a better prognosis, with an increased proportion of antitumor immune cell infiltration. Immunotherapy may improve the prognosis of LUAD patients with a high LINC00987/A2M axis expression.